The major goal of this proposal is to understand the role of p384 mitogen-activated protein kinase (MAPK) in epithelial carcinogenesis. Epithelial cancers, or carcinomas, comprise over 90% of all human cancers. Cutaneous squamous cell carcinoma (SCC) is the second most common cancer in the United States that exhibits poor prognosis in advanced stages. The p38 MAPK family members function in a cell type-specific and cell context-specific manner to regulate cell proliferation, differentiation, survival, inflammation, senescence and transformation. p384 MAPK isoform is highly abundant in cutaneous epithelia, and its expression is increased in human SCC. p384 is also abundantly expressed in innate immune cells known to be involved in maintenance of chronic inflammation associated with epithelial cancer development. Using mice with germline deletion of p384 gene, we recently discovered that p384 is required for de-novo skin tumor development during two-stage chemical carcinogenesis. Here we describe an important phenotype of p384-null mice: these mice are normal phenotypically under homeostatic conditions, but display a significant resistance to chemical carcinogenesis as characterized by delayed tumor onset, reduced frequency, reduced multiplicity and reduced tumor size. Reduced tumor size and late onset suggest growth defects;lower frequency and reduced multiplicity suggest less initiation, or loss of initiated cells during promotion, or ineffective promotion by TPA in p384-null cells. Indeed, our data show that p384 plays an essential role in mediating keratinocyte proliferation during tumor promotion by TPA, and the TPA-mediated induction of key inflammatory and angiogenic mediators, including COX2, VEGF1, and AP1, as well as activation of oncogenic transcription factor Stat3, are abrogated in p384-null setting. Lastly, given the global nature of p384 ablation, p384 may act non cell autonomously, for instance, in the innate immune system, known for its promoting role in skin carcinogenesis. Our additional preliminary data, obtained since the previous review of this application, show that oncogenic ras-transformed p384-null keratinocytes formed smaller skin tumors in orthotopic skin grafts to p384 intact hosts, compared with transformed wild-type keratinocytes. Despite identical latency intervals and similar tumor kinetics during initial weeks after grafting in both genotypes, tumors formed by p384-null keratinocytes exhibited a marked predisposition to regress in size at a late stage of tumor progression. Furthermore, p384-null grafts displayed a reduced rate of malignant conversion compared with their wild-type counterparts. The letter finding suggests that endogenous keratinocyte p384 functions in concert with oncogenic ras to promote malignant conversion of benign tumor cells. Together, the data from chemical carcinogenesis studies, using global p384 knockout mice, and the results from skin graft studies using oncogenic ras-transformed p384-null keratinocytes, support our hypothesis that p384 regulates multiple stages of epithelial carcinogenesis both in a keratinocyte cell autonomous manner, by regulating survival and proliferation of neoplastic epithelial cells, and in a non- cell autonomous manner, by mediating tumor promoting signals emanating from the immune (myeloid) cell component of tumor microenvironment. Using mouse strains we generated to test this hypothesis, we propose to: 1) Determine keratinocyte-specific and immune (myeloid) cell-specific effects of p384 loss on epithelial carcinogenesis by using conditional tissue-specific p384 gene targeting via the Cre-lox system. 2) Determine the impact of p384 loss on distinct stages of tumor development by using epidermal-specific silencing of p384 gene in a temporally regulated fashion, by means of crossing p384flox mice to transgenic mice in which a tamoxifen-regulated CreERT recombinase is expressed under the control of keratinocyte-specific human keratin 14 promoter. The molecular mechanisms underlying p384-dependent contributions to epithelial carcinogenesis will be explored using biochemical and cell culture methods and confirmed in vivo. We are optimistic that the completion of the proposed studies will result in a better mechanistic understanding of the p384 role in epithelial carcinogenesis and its potential as an anti-cancer therapeutic target.

Public Health Relevance

Our recent findings demonstrate a fundamental in vivo importance of p384 mitogen-activated protein kinase signaling in epithelial carcinogenesis. Our proposed research will build on these findings and determine: i) how p384 signaling contributes to specific stages of epithelial carcinogenesis, including initiation, promotion, and malignant progression;and ii) whether p384 promotes tumorigenesis by means of keratinocyte cell autonomous or non cell autonomous mechanisms, or both. Understanding the mechanisms underlying cell-type-specific and tumor stage-specific functions of this newly discovered mediator of epithelial carcinogenesis will have direct and important implications for our understanding of the disease process and will potentially provide an improved strategy for cancer therapy.